Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3′ UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5′ UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.

The RNA-binding protein Sex-lethal (Sxl) is an important post-transcriptional regulator of sex determination and dosage compensation in female Drosophila. To prevent the assembly of the MSL dosage compensation complex in female flies, Sxl acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It uses two distinct and mutually reinforcing blocks to translation that operate on the 5 ′ ′ ′ ′ ′ and 3 ′ ′ ′ ′ ′ untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5 ′ ′ ′ ′ ′ UTR-mediated translational control involves an upstream open reading frame, 3 ′ ′ ′ ′ ′ UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr), which is recruited to the transcript by Sxl. We have identified the protein Sister-of-Sex-lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Both proteins have a comparable RNA-binding specificity and can associate with uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5 ′ ′ ′ ′ ′ UTR of msl-2. However, Ssx is inactive in 3 ′ ′ ′ ′ ′ UTR-mediated regulation, as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain of function and repression via the 3 ′ ′ ′ ′ ′ UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.